Purification and properties of human serum dopamine-β -hydroxylase

1. Two different molecular forms of dopamine-beta-hydroxylase were isolated from human serum; a major component (Peak I enzyme) with a molecular weight of 368000 and with a higher specific activity and a minor component (Peak II enzyme) with a molecular weight of 188000 and with a lower specific activity. 2. Both forms require ascorbic acid for the activity, and are stimulated by fumarate. Addition of N-ethylmaleimide or copper also increased the activity. The optimal pH of both forms in the presence of 20mM tyramine as substrate is 5.0. 3. Km values toward tyramine of Peak I enzyme and Peak II enzyme were 1.67 mM and 14.2 mM respectively. 4. Both Peak I enzyme and Peak II enzyme are glycoprotein.     

Effect of indole-3-acetic acid on cell wall loosening: Changes in mechanical properties and noncellulosic glucose content of Avena coleoptile cell wall

The effect of indole-3-acetic acid on cell wall loosening andchemical modifications of noncellulosic components of the cellwall in Avena coleoptile segments was studied and the followingresults were obtained. (1) Auxin decreased both the minimum stress-relaxation time(T_o) and the noncellulosic glucose content of the cell wall. (2) Decreases were observed in the absence or presence of mannitolsolution at concentrations lower than 0.20 M which osmoticallysuppressed auxin-induced extension, while at concentrationshigher than 0.25 M, there was little auxin effect, indicatingthat it is turgor-dependent. (3) The decrease in T_o of the cell wall and that in the noncellulosicglucose content caused by auxin in the presence of mannitolsolutions of various concentrations paralleled each other (thecorrelation coefficient was 0.897). (4) Both decreases in T_o and glucose content caused by auxinwere inhibited by nojirimycin (5-amino-5-deoxy-D-glucopyranose)in the presence of mannitol. The results suggest that auxin-induced cell wall loosening iscaused by the degradation of noncellulosic rß-glucanin the cell wall. (Received December 24, 1976; )     

Auxin-indnced changes in barley coleoptile cell wall composition

Auxin induces extension growth of barley coleoptile segments,causing cell extension and cell wall loosening represented bya change in mechanical properties of the cell wall. This responsedecreased after the segments were starved for more than 12 hrin buffer solution. Auxin decreased the noncellulosic glucosecontent of the cell wall of the segments starved for 0 and 6hr, but very little that of segments starved for 12 and 18 hr.The contents of arabinose, xylose and galactose, among noncellulosicpolysaccharides, and -cellulose of the cell wall increased duringthe starvation, but auxin did not affect them. The auxin-induceddecrease in glucose content was inhibited by nojirimycin, apotent inhibitor of ß-glucanase, which inhibited auxin-inducedextension and changes in mechanical properties of the cell wall,suggesting that cell wall loosening, and thus cell extension,resulted from partial degradation of ß-glucan of thecell wall. (Received April 20, 1978; )     

Histidine-containing cyclic dipeptides as catalysts in the hydrolysis of carbonic acid p-nitrophenyl esters

A histidine-containing cyclic dipeptide, cyclo(D -Leu-L -His), was almost 20 times as efficient a catalyst as imidazole in the hydrolysis of p-nitrophenyl laurate. The effect of dioxane on the hydrolysis showed that hydrophobic interaction between the cyclic dipeptide and the ester is very important. This reaction obeyed the Michaelis-Menten kinetics, and the Michaelis constant K_m was as low as 9.98 × 10^?5M. Since the linear dipeptide having D -Leu-L -His sequence was nearly inactive in the hydrolysis, the functional groups of cyclo(D -Leu-L -His) in a specific arrangement held by the rigid backbone must have cooperated in the fast hydrolysis. Very weak catalysis by the diasteremeric cyclic dipeptide, cyclo(L -Leu-L -His), in the hydrolysis supported the above view.     

Biopterin cofactor and monoamine-synthesizing monooxygenases

The activities of three pterin-requiring monooxygenases, phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase, are regulated by the level of the pterin cofactor, $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$, which is synthesized from guanosine triphosphate (GTP). Since tyrosine hydroxylase or tryptophan hydroxylase is the rate-limiting enzyme for the biosynthesis of catecholamines (dopamine, norepinephrine and epinephrine) or serotonin in monoaminergic neurons, biosynthesis of tetrahydrobiopterin from GTP may also regulate the tissue level of monoamine transmitters. Recent evidences indicate that biosynthesis of tetrahydrobiopterin and that of biogenic monoamines may be regulated each other.     

Tryptophan hydroxylase system in brain tissue slices assayed by high-performance liquid chromatography

A new method was developed to study the unsupplemented tryptophan hydroxylase system in brain tissue slices from the raphe nuclei of the rat by high-performance liquid chromatography (HPLC) with fluorescence detection. Tryptophan hydroxylase activity was measured by determining 5-hydroxytryptophan (5-HTP) accumulation in raphe nuclei slices containing all of the enzyme system (the hydroxylase, tetrahydrobiopterin, and dihydropteridine reductase) in the presence of NSD-1055 (an inhibitor of aromatic l-amino acid decarboxylase). An optimum temperature was observed at 25°C and the reaction progressed linearly for 60 min. The hydroxylation of tryptophan was maximal by the addition of 0.2 mM tryptophan in the medium. A maximum 1.5-fold activation was shown at 0.2 mM 6-methyltetrahydropterin in the presence of 10 mM dithiothreitol. Dithiothreitol alone did not affect the activity. A 1.5-fold activation was observed when incubation was carried out under gas phase of 95% oxygen and 5% CO₂ instead of air. The activity was inhibited by 75% at 10^?4 M p-chlorophenylalanine. Both A-23187, a calcium ionophore, and dibutyryl cyclic AMP (DBc-AMP) stimulated the hydroxylation of tryptophan. The activation by A-23187 plus DBc-AMP was more than additive, suggesting the two activating mechanisms by Ca²⁺ and cyclic AMP may be operating synergistically.     

Serotonin in neuroblastoma × glioma NG108-15 hybrid cells

Mouse neuroblastoma × rat glioma NG108-15 hybrid cells contain a considerable amount of serotonin, and possess small but significant tryptophan hydroxylase activity. The results suggest that NG108-15 hybrid cells are serotonergic, in addition to the known cholinergic property.     

Comparison between the gelsolin and adseverin domain structure.

Adseverin (74-kDa protein, scinderin) is a calcium- and phospholipid-modulated actin-binding protein that promotes actin polymerization, severs actin filaments, and caps the barbed end of the actin filament, with its NH2-terminal half retaining these properties (Sakurai, T., Kurokawa, H., and Nonomura, Y. (1991) J. Biol. Chem. 266, 4581-4585). Further proteolysis of this NH2-terminal half generated five fragments, and two of them (Mr 15,000 and 31,000) showed Ca(2+)-dependent binding to monomeric actin. The Mr 31,000 fragment especially caused actin filament fragmentation, although its severing activity was also inhibited by several acidic phospholipids as was found in adseverin and its NH2-terminal half. Amino acid sequencing demonstrated that the two fragments' NH2 terminus were blocked in the same manner as the NH2 terminus of adseverin, and thus these two fragments are possibly located at the NH2-terminal of the adseverin molecule. This would then indicate that NH2-terminal fragments had a Ca(2+)-sensitive actin-binding function that relates to actin severing. The other two fragments' NH2-terminal sequencing showed a similar homology to the amino acid sequences of gelsolin and villin. Based on these observations, we propose that adseverin has a functional domain structure similar to that of the gelsolin and villin core.     

Inhibitory effect of calcium-binding protein regucalcin on Ca2(+)-activated DNA fragmentation in rat liver nuclei

Incubation of isolated rat liver nuclei with ATP, NAD+, and micromolar Ca2+ concentrations of various metal ions resulted in extensive DNA hydrolysis. Half-maximal activity occurred with 1.0 microM Ca2+ added, and saturation of the process was observed with 10 microM Ca2+. The Ca2+ (10 microM)-activated DNA fragmentation was inhibited by the presence of Ca2(+)-binding protein regucalcin isolated from rat liver cytosol. The inhibitory effect of regucalcin was complete at 0.5 microM. At 25 microM Ca2+ added, such an effect of regucalcin (1.0 microM) was not seen. Regucalcin also inhibited Ca2(+)-activated DNA fragmentation in the presence of calmodulin (10 and 20 micrograms). The results show that regucalcin can inhibit the Ca2(+)-activated DNA fragmentation due to binding the metal, suggesting a role in regulation of liver nuclear functions.     

Recognition of a 56 kDa protein in partially purified rat hepatic nuclear thyroid hormone receptor by anti-human c-erb A beta antibody.

Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared. The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting. Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction. Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band. These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor.